PROJECT SUMMARY/ABSTRACT Exclusive breastfeeding for at least 4 mo is considered the optimal form of nutrition for most newborns, yet many women experience substantial roadblocks in meeting this goal. One of the most common reason for lactation cessation is mastitis (inflammation of the breast). Mastitis is also a significant concern for the US dairy industry because, not only does it represent a significant challenge to animal welfare, but it also decreases milk production and is the most common reason animals are treated with antibiotics. Despite decades of research, mastitis prevention and treatment are poorly understood in both species. One reason for this is that, although mastitis has historically been attributed to the presence of bacterial pathogens in the mammary gland, this dogma is now known to be incorrect. Modern advances in the use of DNA sequencing (rather than needing to grow bacteria in culture media) have resulted in a paradigm shift in this regard such that researchers now know that all milk produced by both healthy and mastitic cows and women contains a rich community of microbes. Experts now believe that a dysbiosis in these microbes or a shift in their metabolism causes mammary inflammation. Consequently, we must now re-examine everything we thought we knew about the etiology and risk factors for this disease. Fundamental to filling this research gap is the rigorous characterization of the microbiome in milk produced by healthy and mastitic women and cows; and identification of microbial community ?fingerprints? and metabolomic modifiers, thereof, that alter risk of mammary inflammation. In addition, understanding the similarities and dissimilarities in mammary inflammation between cows and women will help researchers understand whether the former can be used as a model for the latter (and vice versa). The overall objectives for this project are to 1) compare and contrast the milk microbiome, its functionality, immune parameters, and inflammatory markers in healthy and mastitic women and cows, and 2) identify milk microbial profiles and their functionality related to risk for mammary inflammation. Our central hypotheses are that 1) mammary inflammation in both species is associated with shifts in microbes and their function, concentrations of selected markers of immunity and mammary inflammation, and that 2) there exist detectable milk microbiome patterns (or functions) that predispose some women and cows to increased risk of inflammation, and these patterns are related to (and perhaps modified by) environmental and behavioral parameters, some of which are currently considered risk factors for mastitis. To test these hypotheses, we will compare healthy and mastitic cows and women (case-control design) during the first 6 wk postpartum (longitudinal, repeated-measures design during a high-risk period in both species). Importantly, we will utilize a multi-omics approach and machine learning to understand complex relationships within and between species. The results of this work will lead to better understanding of how the microbiology of the lactating mammary gland is related to mammary inflammation and will lay the groundwork for future studies to determine how this disease can be prevented and treated.